Polydopamine-Modified Metal–Organic Frameworks, NH2-Fe-MIL-101, as pH-Sensitive Nanocarriers for Controlled Pesticide Release

Recently, metal–organic frameworks (MOFs) have become a dazzling star among porous materials used in many fields. Considering their intriguing features, MOFs have great prospects for application in the field of sustainable agriculture, especially as versatile pesticide-delivery vehicles. However, the study of MOF-based platforms for controlled pesticide release has just begun. Controlled pesticide release responsive to environmental stimuli is highly desirable for decreased agrochemical input, improved control efficacy and diminished adverse effects. In this work, simple, octahedral, iron-based MOFs (NH2-Fe-MIL-101) were synthesized through a microwave-assisted solvothermal method using Fe3+ as the node and 2-aminoterephthalic acid as the organic ligand. Diniconazole (Dini), as a model fungicide, was loaded into NH2-Fe-MIL-101 to afford Dini@NH2-Fe-MIL-101 with a satisfactory loading content of 28.1%. The subsequent polydopamine (PDA) modification could endow Dini with pH-sensitive release patterns. The release of Dini from PDA@Dini@NH2-Fe-MIL-101 was much faster in an acidic medium compared to that in neutral and basic media. Moreover, Dini@NH2-Fe-MIL-101 and PDA@Dini@NH2-Fe-MIL-101 displayed good bioactivities against the pathogenic fungus causing wheat head scab (Fusarium graminearum). This research sought to reveal the feasibility of versatile MOFs as a pesticide-delivery platform in sustainable crop protection.


Introduction
Metal-organic frameworks (MOFs) are crystalline porous materials consisting of organic ligands coordinated to metal centers, and are recent innovations in the field of material chemistry [1,2]. MOFs have become a dazzling star among porous materials by virtue of their superior properties and promising applications in gas storage and separation [3,4], energy conversion and storage [5,6], water harvesting and splitting [7,8], heterogeneous catalysis [9,10], chemical sensors [11,12], environmental remediation [13,14], cancer therapy, drug delivery [15,16], etc. Considering their excellent performance, MOFs have great potential for application in the field of sustainable agriculture,

Synthesis of NH 2 -Fe-MIL-101 Nanocrystals
The synthesis of NH 2 -Fe-MIL-101 nanocrystals used a microwave irradiation method according to the procedure reported previously with a little modification [45]. Briefly, approximately 760.5 mg of H 2 ATA (4.2 mmol) and 2268 mg of FeCl 3 ·6H 2 O (8.4 mmol) were dissolved in 210 mL of deionized water in a 500 mL round-bottom flask. The mixture was thereafter transferred into a Teflon-lined stainless autoclave, sealed and placed in a microwave oven (XH-800G). The autoclave was heated at 100 • C for 4 h by microwave irradiation at 400 W. The obtained NH 2 -Fe-MIL-101 was recovered by centrifugation at 10,000 rpm for 10 min. To remove the free acid, the nanocrystals were washed with fresh ethanol 3 times and dried under vacuum at 80 • C for further characterization and analysis.

Preparation of Dini@NH 2 -Fe-MIL-101 Nanocrystals
Diniconazole was loaded into NH 2 -Fe-MIL-101 nanocrystals by a physical adsorption method. Generally, about 30 mg of Dini and 30 mg of NH 2 -Fe-MIL-101 nanocrystals were weighed in a 10 mL plastic centrifuge tube, and then, 1 mL of dichloromethane was added. Subsequently, the suspension was sealed and stirred at room temperature for 6 h. Diniconazole-loaded NH 2 -Fe-MIL-101 (denoted as Dini@NH 2 -Fe-MIL-101) was collected by centrifugation (10,000 rpm, 10 min) and drying at 50 • C.
To determine the loading content and encapsulation efficiency of Dini, approximately 5 mg of the prepared Dini@NH 2 -Fe-MIL-101 nanocrystals were suspended in 50 mL of methanol and extracted by ultrasonication for 3 h. Then, the concentration of the supernatant was measured by high-performance liquid chromatography (HPLC, 1200-DAD (Diode Array Detector), Agilent, Santa Clara, CA, USA). The HPLC operating conditions were as follows: ZORBAX SB-C 18 reversed-phase column (5 µm, 4.6 × 150 mm); column temperature, 25 • C; mobile phase (methanol/0.1% formic acid aqueous solution (V/V) = 80:20); flow rate, 1.0 mL/min; injection volume, 5 µL; and detection wavelength, 220 nm. The loading content and encapsulation efficiency were calculated by the formulas reported by us [29].

Preparation of Dopamine-Coated Dini@NH 2 -Fe-MIL-101 Nanocrystals
Dopamine-coated Dini@NH 2 -Fe-MIL-101 nanocrystals were prepared according to a previous procedure with a little modification [46]. Briefly, 720 mg of the as-prepared Dini@NH 2 -Fe-MIL-101 and 720 mg of dopamine hydrochloride were dispersed in 360 mL of Tris buffer solution (10 mM, pH = 8.5). The mixture was stirred at room temperature for 24 h. Afterwards, the resulting solid was separated by centrifugation (10,000 rpm, 10 min), washed with water and dried in an oven at 50 • C for 6 h to obtain the dopamine-coated Dini@NH 2 -Fe-MIL-101 (denoted as PDA@Dini@NH 2 -Fe-MIL-101).

Sample Characterization
The surface morphology of as-prepared nanoparticles was observed by scanning electron microscopy (SEM, FEI Quanta Q400, Eindhoven, Netherlands, operated at 20 kV). Powder X-ray diffraction analysis (XRD) was performed on a Bruker D8 Advance X-ray diffractometer (Bruker, Nanomaterials 2020, 10, 2000 4 of 13 Karlsruhe, Germany) with Cu Kα radiation (λ = 0.15418 nm). Data were collected in 2 theta of 5-30 • with a step size of 0.02 • at a scanning rate of 0.1 • /s.
The elemental compositions of the samples were analyzed by X-ray photoelectron spectroscopy (XPS, Kratos Ltd., Manchester, UK) on a photoelectron spectrometer (ESCALab 250Xi, Thermo Fisher Scientific, Waltham, MA, USA) with 150 W monochromatic Al Kα radiation (1486.6 eV, 500 µm spot size) as the excitation source. The binding energies were calibrated by the C1s peak of the surface carbon at 284.8 eV. Energy-dispersive X-ray spectroscopy (EDS) mapping was further used to confirm the elemental composition.
The nitrogen adsorption/desorption isotherms and pore structure of the samples were studied with a specific surface area and pore size analyzer (TriStarII 3020, Micromeritics Instruments Corp, Norcross, GA, USA) at 77 K. The samples were degassed at 10 −3 Torr and 120 • C for 6 h. The chemical structures of the samples were studied with a Fourier transform infrared spectrophotometer (FT-IR, Nicolet 6700, Thermo Fisher Scientific, Waltham, MA, USA) with a potassium bromide pellet. Thermogravimetric analyses (TGAs) were carried out using a PerkinElmer Pyris Diamond (Woodland, CA, USA) from 30 to 550 • C at 10 • C/min under a N 2 atmosphere.

In Vitro Release of Dini
The controlled characteristics of Dini release from Dini@NH 2 -Fe-MIL-101 and PDA@Dini@NH 2 -Fe-MIL-101 were studied by a dialysis method in release medium containing phosphate buffered saline (PBS), ethanol and Tween-80 emulsifier (70:29.5:0.5, v/v/v). The pH-responsive release characteristics were studied in the release medium with different pH values (3.1, 7.0 and 10.3). Approximately 20 mg of pesticide-loaded nanoparticles were weighed in a dialysis bag (MW: 8000-14,000) with 5 mL of release medium. Then, the sealed dialysis bag was immersed in plastic bottles containing 195 mL of release medium and placed on a shaker with a speed of 100 rpm at 25 • C. At designated time intervals, 0.8 mL of the mixture was taken out for HPLC analysis. All the treatment was repeated three times. The accumulative diniconazole released was calculated according to our previous report [29].

Bioactivity Studies
The bioactivities of Dini@NH 2 -Fe-MIL-101 and PDA@Dini@NH 2 -Fe-MIL-101 were studied by the mycelium growth rate method. In this work, the wheat head scab fungus (F. graminearum) was selected as the tested fungus. A mycelial disc with a diameter of 5 mm was inoculated on potato dextrose agar plates. Before inoculation, the sterile molten potato dextrose agar was treated with diniconazole technical concentrate (TC), Dini@NH 2 -Fe-MIL-101 or PDA@Dini@NH 2 -Fe-MIL-101 under two different active-ingredient concentrations of 1 and 5 mg/L. Meanwhile, the bioactivities of the blank carrier for NH 2 -Fe-MIL-101 and control check (CK) without any treatment were also analyzed. Each treatment was repeated five times. After 4 days of incubation at 25 • C, the colony diameter was measured by the cross method and the biological activity is expressed as the percentage of inhibition (%), which was calculated as equal to (colony diameter of control -colony diameter of treatment)/(colony diameter of control -diameter of mycelial discs) × 100.

Statistical Analysis
One-way analysis of variance (ANOVA) and Duncan's multiple range tests were performed on the data using the SPSS 10.0 software (SPSS, Chicago, IL, USA). The confidence intervals used in this study were based on 95% (p < 0.05). All data are plotted as mean ± standard error.

Preparation and Characterization of Nanoparticles
Metal-organic frameworks (MOFs) are a class of crystalline micro-mesoporous hybrid materials. Recently, they have shown potential applications in pesticide-delivery systems due to their high specific surface area and uniform-but-tunable cavities. In the current study, we prepared amino-modified MOFs (NH 2 -Fe-MIL-101) through a rapid microwave-assisted solvothermal synthesis method according to the procedure reported by Horcajada et al. [45]. In fact, most of the references reported the preparation of this MOF by the solvothermal method using conventional heating and dimethyl formamide (DMF) as a solvent, which is adapted from the procedure reported by Bauer et al. [47]. However, when microwave irradiation was used, water instead of DMF was used as the solvent, which is environmentally friendly. Moreover, the reaction time was remarkably shortened from 24 h to 4 h. Thus, a microwave-assisted solvothermal method was adopted to prepare NH 2 -Fe-MIL-101, which was used as a carrier for loading the pesticide diniconazole to afford Dini@NH 2 -Fe-MIL-101. The PDA was thereafter coated on the surface of Dini@NH 2 -Fe-MIL-101 to obtain PDA@Dini@NH 2 -Fe-MIL-101.
The morphology of the as-prepared nanoparticles was observed using SEM. The SEM micrographs showed that NH 2 -Fe-MIL-101 (Figure 1a Figure 3. In the present study, the NH 2 -Fe-MIL-101 was prepared using microwave irradiation. The instantaneous microwave energy causes a reduction in crystallinity [48]. As a result, NH 2 -Fe-MIL-101 shows weaker and fewer diffraction peaks that are consistent with those prepared with a similar method [48]. However, when the conventional solvothermal method was used, more and more-intense diffraction peaks were observed [49,50]. After the loading of Dini, new peaks were found, which were possibly attributable to a Dini salt or some contamination that is washed away after the PDA coating. Successful PDA coating further led to a loss of crystallinity.           The loading content was also measured by an HPLC method, and the result was determined to be 28.1%, which was largely consistent with TGA result. The porous structures of the as-prepared MOFs were examined according to the Brunauer-Emmett-Teller (BET) specific surface area and Barrett-Joyner-Halenda (BJH) pore size and volume analyses. Figure 6 shows the N 2 adsorption-desorption isotherms of NH 2 -Fe-MIL-101, Dini@NH 2 -Fe-MIL-101 and PDA@Dini@NH 2 -Fe-MIL-101. Table 1 summarizes the values of the BET specific surface area (S BET ), the total pore volume (V t ) and the BJH pore diameter (D BJH ) of the samples. After the loading of Dini, the pores of NH 2 -Fe-MIL-101 were filled, and the S BET and V t were reduced from 953.9 to 449.8 m 2 /g and from 0.77 to 0.34 cm 3 /g, respectively, suggesting that most of the micropores were occupied by the Dini molecules. The pores of NH 2 -Fe-MIL-101 were further filled Nanomaterials 2020, 10, 2000 8 of 13 after PDA coating, and the S BET and V t were reduced from 449.8 to 13.0 m 2 /g and from 0.34 to 0.05 cm 3 /g, respectively, suggesting that PDA was successfully coated on the surface of Dini@NH 2 -Fe-MIL-101.

Loading of Dini into NH 2 -Fe-MIL-101 Nanoparticles
The loading content (LC) and encapsulation efficiency (EE) of Dini were optimized by adjusting the solvent and pesticide-carrier mass ratio; the results under various conditions are summarized in Table 2 for the loading test. Under the pesticide-carrier mass ratio of 1:1, when different solvents including acetone, methanol and dichloromethane were used, the LC was 24.9%, 24.3% and 28.1%, respectively. As the solvent would affect the LC, dichloromethane was selected as the solvent to load Dini for further optimization. As expected, the LC increased with an increasing pesticide-carrier ratio, possibly because of the higher Dini concentration, which promotes the carrier's adsorption of pesticide molecules. However, the EE gradually decreased. Considering the LC and EE together, the large-scale preparation of Dini@NH 2 -Fe-MIL-101 samples was performed with a pesticide-carrier ratio of 1:1 for sample characterization, PDA modification, controlled release and bioactivity assays. After PDA coating, the LC was determined to be 14.7% due to the introduction of PDA.

pH-Sensitive Release
Polydopamine microcapsules and PDA-modified materials have been reported for controlled pesticide release because of the pH-responsive characteristic of PDA [30,51,52].   The self-polymerization of dopamine on the surface of NH 2 -Fe-MIL-101 occurs in neutral and basic media to form an adherent polymer coating [30]. The PDA coating could block the pores and confine Dini molecules inside the pores of NH 2 -Fe-MIL-101 in neutral and basic conditions, which is definitely beneficial for avoiding initial burst release [41]. In acidic media, however, the PDA coating might be partially peeled off from the surface of NH 2 -Fe-MIL-101, resulting in a faster release compared to that in neutral or basic media.

Bioassay of Dini-Loaded Nanoparticles
The fungicidal activities of Dini@NH 2 -Fe-MIL-101 and PDA@Dini@NH 2 -Fe-MIL-101 were determined by the mycelium growth rate method. The control efficiencies for wheat head scab (F. graminearum) at two different concentrations, 1 and 5 mg/L, are presented in Figure 8a, and the images of colonies are shown in Figure 8b. The bioactivities of the blank carriers of NH 2 -Fe-MIL-101 and Dini technical concentrate (TC) were also tested as controls. After 4 days of incubation at 25 • C, the inhibition by Dini TC at concentrations of 1 and 5 mg/L was found to be 43% and 83%, respectively. The corresponding inhibition by Dini@NH 2 -Fe-MIL-101 and PDA@Dini@NH 2 -Fe-MIL-101 was (42% and 80%) and (44% and 83%), respectively. The findings clearly indicate that Dini@NH 2 -Fe-MIL-101 and PDA@Dini@NH 2 -Fe-MIL-101 have fungicidal bioactivity against F. graminearum that is comparable to that of Dini TC.  In the present study, the testing of the fungicidal activity against F. graminearum by the plate method was mainly to demonstrate the effectiveness of the as-prepared nano-delivery system, which could not clearly explain the effect of different pHs on controlled release. However, the concept and developed method for controlled pesticide release will find wide application in agricultural practice. The optimal way is to perform the field trails under real application scenarios to demonstrate the relationship between bioactivity and controlled release; this will be the future direction of our research.

Conclusions
In this study, porous NH 2 -Fe-MIL-101 was synthesized through a rapid microwave-assisted solvothermal synthesis using Fe 3+ as the node and H 2 ATA as the organic ligand. Diniconazole, as a model fungicide, was loaded into NH 2 -Fe-MIL-101 by a physical absorption method. Under the optimized conditions of a mass ratio of the pesticide to carrier of 1:1 and using dichloromethane as the solvent, the LC and EE were 28.06% and 40.75%, respectively. The subsequent PDA modification could endow PDA@Dini@NH 2 -Fe-MIL-101 with pH-sensitive release patterns. The release of Dini from PDA@Dini@NH 2 -Fe-MIL-101 was much faster in an acidic medium than that in neutral and basic media. Compared with Dini TC, Dini@NH 2 -Fe-MIL-101 and PDA@Dini@NH 2 -Fe-MIL-101 displayed comparable fungicidal bioactivity against the pathogenic fungus F. graminearum. This research revealed the feasibility of versatile Fe-MOFs as a pesticide-delivery platform in sustainable crop protection.